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. 2023 Oct 9;220(12):e20230558. doi: 10.1084/jem.20230558

Figure 3.

Figure 3.

Depletion of Foxp3+ Tregs facilitates S. aureus growth and spontaneous inflammation. C57BL/6 mice were colonized with a clinical S. aureus as in Fig. 1 A and euthanized on day 10 for ear skin collection and analysis. (A) Absolute numbers of Foxp3+ Tregs in the skin of PBS and S. aureus–colonized mice on day 10. Mean ± SEM representative of two experiments with five mice per group. (B and C) Percent (B) and number (C) of Ki67+Foxp3+ Tregs in the skin of PBS and S. aureus–colonized mice at day 10. Mean ± SEM representative of two experiments with five mice per group. Numbers in contour plots indicate the percent of cells within the gates. (D) Analysis of ICOS, ST2, CTLA4, and CD25 expression on Foxp3+ Tregs. Representative of two experiments with six mice per group. Numbers in contour plots indicate the percent of cells within the gates. (E) S. aureus colonization protocol in Foxp3+ Treg-depleted or non-depleted Foxp3-DTR mice. Mice were colonized on days 1–4 and euthanized on day 10 for ear skin collection and analysis. Mice received DT on days −1, 2, 4, and 6. (F) CFUs of S. aureus at day 10 from the ear skin of S. aureus–colonized Foxp3-DTR mice depleted (Foxp3+ Treg−) or not (Foxp3+ Treg+) of Foxp3+ Tregs by DT treatment. Representative of two experiments with five mice per group. (G) CFUs of S. aureus at day 10 from the dermis (Der) and epidermis (Epi) ear skin of S. aureus–colonized Foxp3-DTR mice depleted or not of Tregs by DT treatment. Representative of two experiments with five mice per group. (H) CFUs of S. aureus at day 10 from the skin-draining lymph node of S. aureus–colonized Foxp3-DTR mice depleted or not of Tregs by DT treatment. Representative of two experiments with five mice per group. (I) Transepidermal water loss (TEWL) in S. aureus–colonized Foxp3-DTR mice depleted or not of Tregs by DT treatment. Representative of two experiments with four to five mice per group. (J) Ear thickness measurement in Foxp3-DTR mice depleted or not of Tregs by DT treatment, with and without S. aureus colonization. Mean ± SD representative of two experiments with five mice per group. (K) H&E-stained sections of Foxp3+ Treg cells non-depleted (Foxp3+ Treg+) and depleted (Foxp3+ Treg−) Foxp3-DTR mice colonized with S. aureus or non-colonized PBS control mice. Scale bar = 200 μm. (L) Ear thickness measurement in Foxp3-DTR mice depleted of Tregs and colonized with different clinical S. aureus isolates from L. braziliensis patients. Mean ± SD representative of two experiments with five mice per group. (M) CFUs of different clinical S. aureus isolates on day 10 from the ear skin of S. aureus–colonized, Treg non-depleted (Foxp3+ Treg+) and depleted (Foxp3+ Treg−) in Foxp3-DTR mice. Representative of two experiments with four mice per group. *P < 0.05, **P < 0.01, and ***P < 0.001. Significant P values by two-tailed unpaired Student’s t test with Welch’s correction, except L, which is one-way ANOVA with Dunnett’s multiple correction, are indicated in the figure.